Isolation And Characterization Of An Enterococcus Faccalis Phage IME-EF1and Expression Of Its Lysin

Objective The problem of bacterial drug resistance is growing with the increasingly serious abuse of antibiotics. The development of new antibiotic appears to be well behind generation of drug‐resistant bacteria, so it is urgent to find new antimicrobial agents. Because of their high efficient sterilization ability and strict host specificity, phages and their lysins have become one of the promising candidates of new antimicrobials, and the research of them have been on the agenda by many researchers in recent years. Enterococcus faecalis is a kind of conditional pathogenic bacteria which is common in people or animals’ intestinal tract, oral and genital tract, and can cause some illness such as endocarditis, bacteremia, urinary tract infection and meningitis. The diseases caused by multi‐resistant enterococcus faecalis have posed a major threat to human health. For the infections caused by gram positive bacteria in nosocomial infection, enterococcus faecalis causes disease with a rate only second to that caused by staphylococcus aureus.This study is to address the current issues of serious drug resistance of enterococcus faecalis. The multi‐resistant enterococcus faecalis strain EF002was obtained from a local hospital. A phage IME‐EF1was isolated with EF002which can widely crack drug resistant enterococcus. Its biological characteristic was examined and its complete genome sequence was determined. The lysine of IME‐EF1was expressed in E.coli and its cracking spectrum and activity were analyzed. An abdominally infection mouse model was established with which the in vivo lysing activities of phage IME‐EF1and its lysin were studied. Methods Enterococcus faecalis EF002isolated by clinical laboratory was selected as indicator bacteria, plaque test was applied to isolate phage from hospital sewage. The phage titer was measured by the double layer plate culture method. The optimal multiplicity of infection and one step growth curve of IME‐EF1were determined. The morphology of the phage was observed using negative staining transmission electron microscope. The phage genome was extracted using proteinase K/SDS method and assayed with electrophoretic analysis after nucleic acid enzyme digestion. The complete phage genome sequence was determined by the IonTorrent Personal Genome Sequencer (PGM).After PCR amplification and ligeted with eukaryotic expression vector pColdI, the phage lysin gene was cloned and expressed. The lysing activity of this protein and its lysing spectrum was studied using spot method and turbidity method. The expression and specific binding of the lysin to the host bacteria were analyzed by ELISA and Western blotting.Enterococcus faecalis EFOO2with minimum lethal dose was used to infect mice abdomenally. At different time points post infection, the infected mice was treated with the phage IME‐EF1or its lysin and the mortality was recorded to roughly assess the therapeutic effects of the phage and its lysin.Results We successfully isolated a phage named IME‐EF1. It could lyse some Enterococcus faecalis strains isolated from local hospitals. IME‐EF1exhibited a tadpole shape under electron microscope. Its optimal multiplicity of infection was determined to be1, and its incubation period was25min. The outbreak period of IME‐EF1was35min, and its burst size was60pfu as determined by the one‐step growth curve. The lysin of phage IME‐EF1(Lysin‐EF2) was expressed in E.coli, and it could lyse all of our Enterococcus faecalis collections, including two strains resistant to vancomycin. Host binding assay demonstrated that the lysin could specially bind with permissive Enterococcus faecalis strains.The animal experiments showed that the phage IME‐EF1and its lysin Lysin‐EF2has a good therapeutic effect for the infection caused by enterococcus faecalis, and the lysin treatment effect was better than the cognate phage. The timely treatment significantly reduced the mouse mortality.Conclusion As the problem of enterococcus faecalis resistant to antibiotics increases seriously, exploring new treatment other than antibiotic was imperative. The present research results showed that phage and its lysin are feasible in the treatment of bacterial infection related diseases, and have the potential to be used as an alternative to antibiotics.